The present application relates to a process for the improved separation of a hydrophobic organic solution from an aqueous culture medium comprising the steps, provision of an aqueous culture medium comprising a metabolically active cell, contacting of the aqueous culture medium with a hydrophobic organic solution, separation of the hydrophobic organic solution from the aqueous culture medium, the cell having a decreased activity, compared to its wild-type, at least of one enzyme that catalyses one of the reactions of the β-oxidation of fatty acids. The invention furthermore relates to the use of a metabolically active cell that has a decreased activity, compared to its wild-type, of an enzyme that catalyses one of the reactions of the β-oxidation of fatty acids, preferably of an enzyme selected from the group comprising FadA, FadB, FadD, FadL and FadE as well as variants thereof, more preferably FadE, for the improved separation of a hydrophobic organic solution from an aqueous culture medium comprising the metabolically active cell.
A fundamental problem in processes for the production of fine chemicals starting from renewable raw materials instead of fossil fuels consists in converting the product once obtained, which is typically initially present in a large-volume aqueous phase, to a hydrophobic organic phase. This conversion is necessary on the one hand to concentrate a finished intermediate or final product and optionally to make possible the synthetic processing in the following reaction steps in organic solution, and on the other hand to improve the yield of the reaction in the aqueous phase by the removal of the desired product or firstly to make possible the course of the reaction in a technically meaningful context at all, in particular when the presence of the product acts disadvantageously on the reaction progress on account of toxicity to the production strain or an inhibition of a relevant biocatalyst by the product. The direct thermal concentration of the product, frequently present in low concentrations, from the large-volume aqueous solution is generally not expedient.
An example of such a strongly demanded product industrially, which is conventionally produced starting from hydrocarbons contained in petroleum, is 12-aminolauric acid (ALA) or its methyl ester (ALAME). ALA is an important starting product in the production of polymers, for example for the production of piping systems based on Nylon. Conventionally, ALA is produced in low yield starting from fossil raw materials in a process via laurolactam, which is synthesized by trimerization of butadiene, subsequent hydrogenation with formation of cyclododecane, subsequent oxidation to cyclododecanone, reaction with hydroxylamine and subsequent Beckmann rearrangement. A promising new route to the biotechnological production of ALA or ALAME is described in WO 2009/077461. The biotechnological process on which this route is based may be conducted in a two-phase system using a liquid ion exchanger, as is described in EP 11154707.
The workup of a product from an aqueous phase by means of an extraction into a hydrophobic organic phase firstly requires that this product has an adequate tendency to enter into the organic phase in a two-phase system comprising an aqueous, hydrophilic phase and an organic, hydrophobic phase that do not mix, which depends significantly on the physicochemical properties of the respective compound. While compounds with a high content of or consisting exclusively of unsubstituted hydrocarbons enrich mainly in the hydrophobic phase, compounds with a high content of polar groups such as heteroatom-containing functionalities and very particularly compounds with charges are mainly or virtually exclusively present in the aqueous phase, which complicates a transfer to an organic phase.
The partition of a compound in the two-phase system mentioned after adjustment of equilibrium is frequently described with the aid of partition coefficients, for example according to Nernst's equationα=cphase 1/cphase 2.
A special partition coefficient is Kow, also described as the P value, which characterizes the partition equilibrium of a compound between an octanol and an aqueous phase:Kow=P=coctanol/cwater 
A further prerequisite for the workup of the product from the hydrophobic phase consists in that the partition has reached the equilibrium state, which is described by the aforementioned equations, or at least approaches it sufficiently. The adjustment of the equilibrium is determined, inter alia, by the size of the contact area between both phases, a factor that is generally non-limiting in the case of biotechnological processes, since the contact area is already increased by measures such as aeration and the thorough stirring of the aqueous culture medium and the hydrophobic organic solution, which are necessary anyway due to maintenance of high densities of metabolically active cells.
Before such a reaction mixture, in which the hydrophobic organic solution is mainly present in micelles or other subcompartments, can be worked up efficiently, however, a separation of the two phases is necessary. The formation of two phases often takes place spontaneously and without any further action on mixing pure water with a pure organic hydrophobic solvent. However, the separation of an organic hydrophobic solution from a complex aqueous culture medium is less simple because of the many possible component interactions and without technical support, for example centrifugation, the separation may require from several hours to several days.
For the large-scale production of industrially demanded chemical compounds by of biotechnological processes, the process of phase separation may be a factor of considerable importance in the development and application of processes for resource-saving and rapid production and workup of compounds such as ALA. If the product is removed dissolved in a hydrophobic organic solution from a large-volume aqueous phase in a large reactor and subsequently processed, in addition to the parameters relevant for the actual production, such as type and amount of substrates, temperature and oxygen content of the medium, the separation of the hydrophobic solution must be optimized to save resources and to make the entire process as environmentally friendly as possible. Since numerous hydrophobic organic solvents used on a large scale may have a toxic action on organisms used biotechnologically, at least on relatively long contact, it is desirable to shorten the contact between the organism in the aqueous culture medium. By minimizing such contact with solvents, the protection biotechnologically used strains may be protected and release of undesired by-products released during the lysis of cells contaminating or even decomposing the target product may be prevented.